Traumatic brain injury (TBI) is called a silent epidemic. The Centers for Disease Control and Prevention estimate that 1.4 million people sustain TBIs each year, resulting in about 235,000 hospitalizations and 50,000 deaths. Almost half a million TBIs occur in children to 14 years of age. Patients often present with symptoms that are vague and merely suggestive of TBI. A simple, affordable blood test indicating brain injury could significantly impact care—from infants suffering from abuse, to head injuries from sports accidents, car crashes, and soldiers at war.
A group of researchers at Children’s Hospital of Pittsburgh of University of Pittsburgh Medical Center (UPMC) embarked on a research study to evaluate biomarkers in infants in emergency departments at risk of brain injury. The goal is to develop a rapid, affordable test at the point of care that would indicate brain injury. The team is led by Rachel Berger, M.D., an assistant professor of pediatrics at the University of Pittsburgh, School of Medicine.
Using the Axela dotLab® System, the researchers are testing for neuron specific enolase (NSE), a homodimer of the gamma form of enolase. NSE is localized in the cytoplasm of neurons and cells of neuronal origin. Its presence in cerebrospinal fluid and blood is attributed to cell destruction and has shown strong potential as a screening test for traumatic brain injury.
ELISA Test Transfer
Serum NSE levels can be measured with commercial ELISA kits. Though quite sensitive, ELISA is time consuming and best suited for a large number of samples. Although a good research tool, ELISA lacks the reproducibility required of a clinical test and because of the needs for a calibration curve for each sample, it is considered too expensive for clinical care.
For this research study, Axela scientists applied the principles of the ELISA method to develop a dotLab NSE assay and improve the throughput characteristics and robustness of the assay. Based on the NSE ELISA, the Axela dotLab System demonstrated its use as a rapid alternative to ELISA analysis.
Standard ELISA techniques use a pair of monoclonal anti-NSE antibodies that recognize different nonoverlapping epitopes on the antigen. One of the antibodies is designated as a capture antibody and labeled with biotin residues for immobilization onto avidin-coated ELISA plates. NSE from samples binds to the capture antibody on the plate. HRP-tagged detector antibodies are used in the next step. The signal is visualized by appropriate enzymatic reaction and detected by spectrophotometer.
In the dotLab System, affinity reagents (e.g., avidin or amine reactive substrates) are prepatterned on the surface of the inexpensive, disposable dotLab Sensor. For the dotLab NSE assay, biotinylated monoclonal anti-NSE capture antibody (CAb) binds strongly to the avidin surface of the dotLab Sensors that create the diffraction grating. An HRP-conjugated detector monoclonal anti-NSE antibody (DAb) is preincubated with NSE to form a detection complex. The resulting complex binds to the capture antibody on the surface of the sensor through the antigen, as the CAb recognizes a nonoverlapping epitope of the antigen. (Figure 1).
The dotLab System automatically delivers samples and reagents from a standard 96-well plate or bulk containers. Complex matrices including blood and plasma can normally be used directly without labeling or purification. The patterned capture molecules on the dotLab Sensor interact with sample in a 10 µL flow channel.
Diffractive optics technology (dot®) quantitatively measures the change in diffraction as sample molecules bind to the sensor surface in real-time (Figure 2). As sample molecules bind to the sensor surface, diffraction efficiency improves in proportion to size and concentration of the target. A prism molded into the base of the disposable sensor allows operation in total internal reflection mode, effectively eliminating interference and the need for a separate reference channel. Binding is determined quantitatively by monitoring the change in intensity of the diffractive signal.
dotLab NSE Assay
The dotLab NSE assay takes 20 minutes, making it well suited for use in the fast-paced setting of an emergency room. The NSE serum assay has been shown to be sensitive, linear within the working range of concentrations, robust, and rapid. Specifically, the NSE assay developed demonstrated:
• linearity in the range of interest (10–80 ng/mL NSE) (Figure 3),
• a limit of quantitation of 1.9 ng/mL NSE,
• reproducibility within the same system and robust transfer to another operator,
• matrix independence (when PBST buffer was substituted for normal human serum) except when the degradation of analyte occurred, and
• prevention of NSE antigen degradation using a cocktail of protease inhibitors (Figure 4).
Researchers at the Children’s Hospital of Pittsburgh of UPMC are using the dotLab System NSE assay and have plans to add other brain injury biomarkers such as myelin-basic protein in the future. Such a test could help to distinguish whether a fussy or irritable infant simply has a virus or whether the symptoms are the result of a brain injury. A patient with an increased NSE level could then benefit from a CT head scan, a procedure that is not administered lightly since irradiating infants and young adults can be of concern. The NSE test also promises to predict outcome, where a high NSE level may help guide the physician in choosing the right treatment options for their patients.